Alcohol has pervasive impacts on presynaptic functions, yet the mechanisms underlying these considerable impacts are largely unknown. It is likely that these presynaptic mechanisms contribute significantly to the development of alcohol dependence. Identifying the molecular participants responsible for consummating ethanol's presynaptic impact is necessary to develop new targets to fight addiction and recidivism. It is particularly important to define the target of ethanol binding as this may bring about the most complete effect. The long-term goal of this application is to unfold the presynaptic mechanisms for ethanol action. The objective of this proposal is to describe the interaction between ethanol and Munc13-1 and define the effects of this interaction in presynaptic physiology and behavior. Munc13-1 is a conserved presynaptic active zone protein essential for neurotransmitter release in the brain. Preliminary data demonstrate that ethanol binds to the C1 diacylglycerol-binding domain of Munc13-1. A reduction in Dunc13, the Drosophila Unc13 homolog results in flies that are resistant to ethanol and have defects in tolerance and self-administration. The central hypothesis to be tested is that a significant effect of ethanol on nervous system function is due to the binding of ethanol to the Munc13 C1 domain. This hypothesis will be tested in 4 aims. In Aim 1, the atomic structure of ethanol bound to the C1 domain of Munc13-1 will be determined. In Aim 2, the effect of ethanol on Unc13 vesicle fusion will be measured in vitro. In Aim 3, wild type Munc13-1 and Munc13-1 with mutations that reduced ethanol affinity will be used to functionally complement the Dunc13 haploinsufficient behavioral phenotypes. Moreover, the effect of the ethanol-Munc13-1 interaction will be examined using the synapto- pHluorin sensor to image synaptic vesicle release in intoxicated and sober flies. These experiments will determine how ethanol binding to Munc13-1 alters the activity of this protein in vivo. In Aim 4, we determine if heterozygous Munc13-1KO/+ mice, similar to the Drosophila Dunc13 mutants, have defects in ethanol sensitivity and self-administration. The approach is innovative as it applies in a single project the strengths of atomic level resolution, in vitro biochemistry, and in vivo behavior and physiology, to understand the function of an ethanol-effector interaction. This proposal is significant as it will likely provide unequivocal information on the importance of a general mechanism by which ethanol impacts presynaptic function, and how this mechanism is critical in the process required for alcohol dependence. Ultimately, these results will provide precise structural information on where alcohol binds Munc13-1, and how this interaction alters Munc13-1 activity, how this interaction impacts ethanol sensitivity and self-administration. The results will enable the design and validation of small molecule inhibitors that could be used in the development of drugs to fight dependence and recidivism.